U.S. patent application number 11/602835 was filed with the patent office on 2007-03-29 for ubiquitin-specific protease occurring in the brain and dna encoding the same.
Invention is credited to Tatsuo Suzuki, Qing Bao Tian.
Application Number | 20070072269 11/602835 |
Document ID | / |
Family ID | 19146074 |
Filed Date | 2007-03-29 |
United States Patent
Application |
20070072269 |
Kind Code |
A1 |
Suzuki; Tatsuo ; et
al. |
March 29, 2007 |
Ubiquitin-specific protease occurring in the brain and DNA encoding
the same
Abstract
A ubiquitin-specific protease occurring in the brain and a DNA
encoding it, which are useful for research on the molecular
mechanism of the neuroplasticity expression and so on.
Inventors: |
Suzuki; Tatsuo;
(Matsumoto-shi, JP) ; Tian; Qing Bao;
(Matsumoto-shi, JP) |
Correspondence
Address: |
CHOATE, HALL & STEWART LLP
TWO INTERNATIONAL PLACE
BOSTON
MA
02110
US
|
Family ID: |
19146074 |
Appl. No.: |
11/602835 |
Filed: |
November 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10494026 |
Apr 29, 2004 |
7157265 |
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PCT/JP02/11231 |
Oct 29, 2002 |
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11602835 |
Nov 21, 2006 |
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Current U.S.
Class: |
435/69.1 ;
435/226; 435/320.1; 435/325; 530/388.26; 536/23.2 |
Current CPC
Class: |
C07K 16/40 20130101;
C12N 9/6472 20130101 |
Class at
Publication: |
435/069.1 ;
435/226; 435/320.1; 435/325; 530/388.26; 536/023.2 |
International
Class: |
C12N 9/64 20060101
C12N009/64; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C07K 16/40 20060101 C07K016/40 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 29, 2001 |
JP |
2001-330339 |
Claims
1.-9. (canceled)
10. An antibody directed to an isolated ubiquitin-specific protease
that comprises the amino acid sequence set forth in SEQ ID NO: 2;
or an isolated ubiquitin-specific protease that consists of the
amino acid sequence of SEQ ID NO: 2.
11. An antibody directed to a peptide having the amino acid
sequence of the amino acid numbers 1022 to 1036 in the amino acid
sequence of SEQ ID NO: 2.
Description
TECHNICAL FIELD
[0001] The present invention relates to a ubiquitin-specific
protease occurring in the brain and a DNA encoding the same.
BACKGROUND ART
[0002] It is considered that the local protein synthesis using
mRNAs distributed in dendrites plays an important role in
maintaining the already expressed synaptic plasticity. The
synthesis in the postsynaptic region is considered especially
important, and search for mRNAs associated with the postsynaptic
density (PSD) has been carried out (Mol. Brain Res., 72:147-157,
1999). However, functions remain unknown for many of them.
DISCLOSURE OF THE INVENTION
[0003] An object of the present invention is to provide a protein
considered to exist in PSD and a DNA encoding it, which are useful
for research on the molecular mechanism of the neuroplasticity
expression and so forth.
[0004] The inventors of the present invention succeeded in
obtaining a full-length cDNA for one of mRNAs described in Mol.
Brain Res., 72:147-157, 1999, of which functions are unknown, and
elucidating the function of the expression product thereof, and
thus accomplished the present invention.
[0005] The present invention provides the followings. [0006] (1) A
protein of the following (a) or (b): [0007] (a) a protein which has
the amino acid sequence of SEQ ID NO: 2; [0008] (b) a protein which
has an amino acid sequence of SEQ ID NO: 2 including deletion,
substitution or addition of one or several amino acid residues and
has a ubiquitin-specific protease activity. [0009] (2) The protein
according to (1), which has the amino acid sequence of SEQ ID NO:
2. [0010] (3) A DNA which encodes the protein as defined in (1) or
(2). [0011] (4) The DNA according to (3), which has the nucleotide
sequence of the nucleotide numbers 178 to 3285 in the nucleotide
sequence of SEQ ID NO: 1. [0012] (5) A DNA of the following (a) or
(b): [0013] (a) a DNA which has the nucleotide sequence of the
nucleotide numbers 178 to 3285 in the nucleotide sequence of SEQ ID
NO: 1; [0014] (b) a DNA which hybridizes to a DNA having a
nucleotide sequence complementary to the nucleotide sequence of the
nucleotide numbers 178 to 3285 in the nucleotide sequence of SEQ ID
NO: 1 under a stringent condition and encodes a protein having a
ubiquitin-specific protease activity. [0015] (6) A DNA of the
following (a) or (b): [0016] (a) a DNA which has the nucleotide
sequence of the nucleotide numbers 178 to 3285 in the nucleotide
sequence of SEQ ID NO: 1; [0017] (b) a DNA which has a nucleotide
sequence showing a homology of 85% or more with respect to the
nucleotide sequence of the nucleotide numbers 178 to 3285 in the
nucleotide sequence of SEQ ID NO: 1 and encodes a protein having a
ubiquitin-specific protease activity. [0018] (7) A recombinant
vector comprising the DNA as defined in any one of (3) to (6).
[0019] (8) A transformant obtained by transforming a host with the
DNA as defined in any one of (3) to (6). [0020] (9) A method for
producing a ubiquitin-specific protease, which comprises culturing
the transformant as defined in (8) and collecting a
ubiquitin-specific protease expressed by the transformant from
culture. [0021] (10) An antibody directed to the protein as defined
in (1) or (2). [0022] (11) An antibody directed to a peptide having
the amino acid sequence of the amino acid numbers 1022 to 1036 in
the amino acid sequence of SEQ ID NO: 2.
BRIEF EXPLANATION OF THE DRAWING
[0023] FIG. 1 shows (a) a structure of expression product and (b)
detection of ubiquitin-specific protease activity (electrophoresis
photograph).
BEST MODE FOR CARRYING OUT THE INVENTION
[0024] Hereafter, the present invention will be explained in
detail.
[0025] Among the proteins of the present invention, the protein
having the amino acid sequence of SEQ ID NO: 2 is a protein
identified as a ubiquitin-specific protease existing in the brain,
as described in the examples mentioned later. For a protein,
existence of a mutant having the same function is expected, and
such a mutant having the same function can be obtained by suitably
modifying the amino acid sequence of the protein. Therefore, a
protein having an amino acid sequence of SEQ ID NO: 2 including
deletion, substitution or addition of one or several amino acid
residues and having a ubiquitin-specific protease activity also
falls within the scope of the protein of the present invention.
[0026] An amino acid sequence of a protein can be modified by
modifying a DNA encoding the protein with a well-known means such
as the site-specific mutagenesis method and expressing the DNA of
which nucleotide sequence is modified. Furthermore, the
ubiquitin-specific protease activity is an activity of specifically
proteolysing a ubiquitinated substrate protein, and this activity
can be measured by a known method (refer to, for example, EMBO J.,
16:1519-1530, 1997). Therefore, whether a protein has the same
function or not can be easily determined by those skilled in the
art.
[0027] The protein of the present invention preferably has the
amino acid sequence of SEQ ID NO: 2.
[0028] The protein of the present invention may be made into a
fusion protein by fusion with another protein such as glutathione
transferase (GST) or a His-tag.
[0029] The DNA of the present invention is a DNA encoding the
protein of the present invention. An example of the DNA of the
present invention is a DNA having the nucleotide sequence of the
nucleotide numbers 178 to 3285 in the nucleotide sequence of SEQ ID
NO: 1. This DNA is a DNA of which nucleotide sequence was
determined in the examples mentioned later. For a gene, existence
of another gene having a different nucleotide sequence, but
encoding the same product, or an allelic gene encoding a mutant
having the same function is expected, and a gene encoding the same
product or the mutant having the same function can be obtained by
modifying the nucleotide sequence. Therefore, a DNA having a
nucleotide sequence analogous to the nucleotide sequence of the
nucleotide numbers 178 to 3285 in the nucleotide sequence of SEQ ID
NO: 1 and encoding a protein having a ubiquitin-specific protease
activity also falls within the scope of the DNA of the present
invention. Examples of DNA having the analogous nucleotide sequence
include a DNA which hybridizes to a DNA having a nucleotide
sequence complementary to the nucleotide sequence of the nucleotide
numbers 178 to 3285 in the nucleotide sequence of SEQ ID NO: 1
under a stringent condition and a DNA which has a nucleotide
sequence showing a homology of 85% or more with respect to the
nucleotide sequence of the nucleotide numbers 178 to 3285 in the
nucleotide sequence of SEQ ID NO: 1.
[0030] Examples of the stringent condition include the conditions
of hybridization in 2.times. SSC containing 0.1% SDS at 50.degree.
C. and subsequent washing in 2.times.SSC containing 0.1% SDS at
25.degree. C. for 0.5 hour. The washing is preferably washing in
1.times.SSC containing 0.1% SDS at 60.degree. C. for 0.5 hour.
[0031] The homology is represented with a value calculated by using
Genetryx (Software Development Co., Ltd.).
[0032] The DNA having the nucleotide sequence analogous to the
nucleotide sequence of the nucleotide numbers 178 to 3285 in the
nucleotide sequence of SEQ ID NO: 1 can be obtained by subjecting a
DNA having the nucleotide sequence of the nucleotide numbers 178 to
3285 in the nucleotide sequence of SEQ ID NO: 1 or a host harboring
such a DNA to a mutagenesis treatment and selecting a DNA
hybridizing under the aforementioned stringent condition or having
a homology within the above-defined range from the obtained
transformants. The method for measuring the ubiquitin-specific
protease activity is known as described above, and therefore it is
easy for those skilled in the art to select a DNA encoding a
protein having the ubiquitin-specific protease activity from such
DNAs.
[0033] The DNA of the present invention can be obtained in a
conventional manner on the basis of the nucleotide sequence
elucidated herein. For example, it may be synthesized by a chemical
synthesis method or may be obtained by the reverse transcription
PCR method using suitably designed primers from mRNAs prepared from
cells or tissues expressing the ubiquitin-specific protease of the
present invention.
[0034] The vector of the present invention is a recombinant vector
containing the DNA of the present invention. The vector of the
present invention can be obtained by inserting the DNA of the
present invention into a vector in a conventional manner. The
vector to which the DNA of the present invention is inserted is not
particularly limited, and its examples include those usually used
as cloning vectors and those usually used as expression vectors for
mammalian cells. When the vector is used for the purpose of
producing the protein of the present invention, an expression
vector is especially useful.
[0035] The transformant of the present invention is a transformant
obtained by transforming a host with the DNA of the present
invention, and it expresses the protein of the present
invention.
[0036] The host is not particularly limited, and its examples
include animal cells, bacterial cells, yeast cells, insect cells
and so forth. The transformation can be carried out in a
conventional manner, and it is preferably carried out by
introducing the vector of the present invention.
[0037] The production method of the present invention is a method
for producing the protein of the present invention, i.e., a
ubiquitin-specific protease, and it comprises culturing the
transformant of the present invention and collecting a
ubiquitin-specific protease expressed by the transformant from
culture.
[0038] The culturing can be carried out under a condition that
allows the transformant to express the protein of the present
invention, and the collection of the protein of the present
invention from the culture can be attained by a suitable
combination of methods usually used for purification of proteins
such as various chromatography techniques, electrophoresis and gel
filtration. When the protein of the present invention is expressed
as a fusion protein with GST or His-tag, the fusion protein can be
purified by using a glutathione-Sepharose column or
nickel-Sepharose column, respectively.
[0039] The antibody of the present invention is an antibody
directed to the protein of the present invention, and it is
preferably an antibody directed to a peptide having the amino acid
sequence of the amino acid numbers 1022 to 1036 in the amino acid
sequence of SEQ ID NO: 2 (also referred to as the "C-terminus
peptide" hereinafter). In this specification, the term "antibody
directed to" means an antibody that immunologically reacts.
[0040] The antibody of the present invention can be obtained from
an animal immunized with the protein of the present invention
(preferably, the C-terminus peptide). If it is a polyclonal
antibody, it can be prepared from blood serum of the immunized
animal. If it is a monoclonal antibody, it can be prepared by
fusing an antibody-producing cell obtained from the spleen or lymph
node of the immunized animal with a myeloma cell and selecting a
hybridoma that produces an antibody showing strong specificity to
the protein of the present invention (preferably, the C-terminus
peptide).
[0041] As the immunogen, a fragment of the protein of the present
invention obtained according to the production method of the
present invention can be used. Alternatively, an antigen
synthesized on the basis of the aforementioned amino acid sequence
can be used. The antigen may be used as a complex with a carrier
protein. Various linking agents can be used for the preparation of
the complex of the antigen and the carrier protein, and
glutaraldehyde, carbodiimide, maleimide active ester and so forth
can be used. The carrier protein may be selected from those
conventionally used such as bovine serum albumin, thyroglobulin and
hemocyanin, and a method of coupling it in a ratio of 1- to 5-fold
amount is used.
[0042] The animal used for immunization may be mouse, rat, rabbit,
guinea pig, hamster or the like, and inoculation may be carried out
by subcutaneous, intramuscular, intraperitoneal administration or
the like. Upon the administration, the antigen for immunization may
be mixed with complete Freund's adjuvant or incomplete Freund's
adjuvant and then administered. The administration is usually
carried out every 2 weeks to 5 weeks An antibody-producing cell
obtained from the spleen or lymph node of the immunized animal is
fused to a myeloma cell and isolated as a hybridoma. As the myeloma
cell, those derived from mouse, rat, human and so forth are used.
Although it is preferably a myeloma cell derived from the same
species as that from which the antibody-producing cell is derived,
it may be a heterozoic cell.
[0043] The cell fusion can be carried out according to a known
method, for example, the method of Koehler and Milstein (Nature,
256, 495, 1975). As a cell fusion promoting agent, polyethylene
glycol, Sendai virus or the like can be mentioned, and the cell
fusion can be usually carried out by a reaction for about 1 to 10
minutes using polyethylene glycol (average molecular weight: 1000
to 4000) at a concentration of about 20 to 50% at a temperature of
20 to 40.degree. C., preferably 30 to 37.degree. C., usually with a
number ratio of antibody-producing cells and myeloma cells of about
1:1 to 10:1.
[0044] For screening for antibody-producing hybridoma, various
immunochemical methods can be used. Examples thereof include ELISA
(enzyme-linked immunosorbent assay) method using a microplate
coated with the protein of the present invention (preferably, the
C-terminus peptide), EIA (enzyme immunoassay) method using a
microplate coated with an anti-immunoglobulin antibody,
immonoblotting method using a nitrocellulose membrane to which
electrophoresed sample containing the protein of the present
invention (preferably, the C-terminus peptide) is transferred and
so forth.
[0045] From a well thus screened, cloning is further carried out
by, for example, limiting dilution analysis to obtain clones.
Selection and culture of hybridoma are usually carried out with a
medium for animal cells (e.g., RPMI1640) supplemented with HAT
(hypoxanthine, aminopterin and thymidine) and containing 10 to 20%
of bovine fetal serum. The clones obtained as described above can
be transplanted into the abdominal cavity of SCID mouse to which
pristine is preliminarily administered, and after 10 to 14 days,
the ascites containing monoclonal antibodies at a high
concentration can be extracted and used as the raw material for
purification of the antibodies. Moreover, it is also possible to
culture the clones and use the culture broth as the raw material
for purification of the antibodies. For the purification of
monoclonal antibodies, a method known as a purification method of
immunoglobulin can be used, and the purification can be easily
attained by, for example, ammonium sulfate fractionation, PEG
fractionation, ethanol fractionation, use of anion exchanger,
affinity chromatography using the protein of the present invention
(preferably, the C-terminus peptide) or the like. Purification of
polyclonal antibodies from blood serum can also be carried out in a
similar manner.
[0046] As the monoclonal antibody, a fraction of Fab' or Fab in
which Fc' or Fc region is removed, or a polymer thereof may be
used. Moreover, a chimeric antibody or humanized antibody thereof
may also be used.
[0047] By an immunological method using the antibody of the present
invention, the protein of the present invention in a biological
sample can be qualitatively or quantitatively determined. As the
immunological method, known methods such as immunostaining method,
enzyme immunoassay, agglutination method, competitive immunoassay
and sandwich assay can be applied for a biological sample subjected
to a suitable treatment, for example, separation of cells,
extraction etc. as required. The immunostaining method can be
carried out by, for example, the direct method using a labeled
antibody, indirect method using a labeled antibody directed to the
antibody or the like. As the labeling agent, any of known labeling
agents such as fluorescent substances, radioactive substances,
enzymes, metals and dyes can be used.
EXAMPLES
[0048] Hereafter, the present invention will be explained more
specifically with reference to the following examples.
Example 1
Cloning of Full Length Dem 21 cDNA
[0049] The Dem 21 double-stranded cDNA (length: 215 bp) was
obtained as reported in Mol. Brain Res. 72:147-157, 1999. The
obtained cDNA was labeled with digoxigenin (DIG) by the PCR
labeling method to obtain a DIG-labeled Dem 21 cDNA probe.
[0050] In screening libraries prepared from unstimulated rat brains
by using this probe, any positive clone was not obtained.
Therefore, an oligo-(dT) primed cDNA library prepared from a
hippocampus harvested 4 hours after high-frequency stimulation was
screened.
[0051] The oligo-(dT) primed cDNA library prepared from hippocampus
harvested 4 hours after high-frequency stimulation in rats
pretreated with cyclohexamide (Neuron, 14:433-455, 1995) was kindly
provided by Dr. Yamagata of Tokyo Metropolitan Institute for
Neuroscience. This library was plated at a density of
5.times.10.sup.4 plaques per 150-mm plate, and filters (membranes)
on which the plates were duplicated were screened by hybridization
to the DIG-labeled Dem 21 cDNA probe. The hybridization was carried
out overnight at 42.degree. C. in the DIG Easyhyb buffer. The
membranes were washed twice with 2.times.SSC containing 0.1% SDS at
room temperature and then washed with 1.times.SSC containing 0.1%
SDS at 65.degree. C. for 15 minutes. Color development was carried
out according to the instruction of the DIG Chemical Color
Development Kit (Nippon Roche). Candidate plaques were selected,
and re-screening was carried out in a similar manner. Inserts of
positive cDNA clones were excised and confirmed by DNA sequencing.
As a result, two of partially overlapped positive clones with
different lengths, both of which had the probe sequence, were
obtained (18'2 and 17'1). The clone 18'2 contained a poly(A).sup.+
sequence and an insert of 2163 bp having AAATTAAA polyadenylation
signal upstream from the poly(A).sup.+ by 30 bp. The clone 17'1
contained an insert of 4369 bp having the same 3' sequence as that
of the clone 18'2.
[0052] Since the longer clone contained a partial open reading
frame (ORF), 5' RACE was carried out in order to obtain the full
length cDNA of Dem21, to obtain a further 5' sequence.
Specifically, synthesis of a single-stranded cDNA for nMRNA (0.5
.mu.g) prepared from the brain of rats treated with kainic acid by
a reverse transcription reaction, cyclization of the obtained cDNA,
and PCR by using the cyclized cDNA as a template were carried out
twice by using 5' Full Race Core Set and Taq DNA polymerase (both
from Takara Shuzo). In the first 5' RACE, a gene-specific primer
for a reverse transcription reaction (SEQ ID NO: 4) and an
antisense primer and a sense primer for PCR (SEQ ID NOS: 5 and 6),
which were prepared on the basis of the cDNA sequence of 17'1, were
used. In the second 5' RACE, a gene-specific primer for a reverse
transcription reaction (SEQ ID NO: 7) and an antisense primer and a
sense primer for PCR (SEQ ID NOS: 8 and 9), which were prepared on
the basis of the nucleotide sequence obtained by the first 5' RACE,
were used.
[0053] By the first 5' RACE, a 1.0-kb fragment overlapping the 5'
portion of the clone 17'1 was obtained. Furthermore, by the second
5' RACE, a 800-bp fragment overlapping the fragment obtained by the
first 5' RACE was obtained. By combining the cDNA sequence of 17'1
and the sequences obtained by 5' RACE, cDNA of 5738 bp was
obtained. The nucleotide sequence thereof is shown in SEQ ID NO: 1.
This 5738 bp sequence contained ORF of 3111 bp. As described above,
the poly(A).sup.+ tail was preceded by a consensus AAATTAAA
polyadenylation signal. The 5' portion of 178 nucleotides from the
5' end of the putative initiation codon was rich in GC, and was
consistent with the 5' untranslation region. The flanking
nucleotide sequence around the putative initiation codon was
consistent with the Kozak consensus sequence (J. Biol. Chem.,
266:19867-19870, 1991) with Gs at -3 and +4. The presence of a
CT-rich sequence and an in-frame stop codon upstream of the
putative initiation codon further verified that the ATG was a real
initiation codon. From the above, it was found that the obtained
cDNA of 5738 bp contained the full length cDNA of Dem 21, and the
fragment of 215 bp reported in Mol. Brain Res., 72:147-157, 1999
was the 3' end of the full length cDNA having the poly
(A).sup.+.
[0054] A homology search of the deduced amino acid sequence of the
full length Dem 21 cDNA clone (SEQ ID NO: 2) with the DDBJ nr
database did not show an identical protein or any protein species
with strong similarities. However, the deduced protein contained
many domains in which Cys box, His box, Asp box, KRF box etc.
observed in the ubiquitin-specific protease (USP) were conserved.
On the basis of this characteristic, Dem 21 was considered to be a
novel member of the USP family enzymes, and therefore Dem 21 was
named synaptic USP (synUSP). Among the known USPs, synUSP is the
most similar to HAUSP (herpesvirus related ubiquitin-specific
protease), which is named USP7 according to the nomenclature
proposed by the Human Genome Organization (HUGO) Nomenclature
Committee (http://www.gene.ucl.ac.uk/nomenclature). Between two of
these proteins, there was 28% identity and 40% similarity for 413
amino acid residues.
[0055] synUSP encodes a protein of 1036 amino acid residues and has
a predicted molecular mass of 118.78 kDa and pI of 5.83. In the
database search, besides the USP active site domain, a leucine
zipper domain was detected in the carboxyl terminus region. Since
the leucine zipper domain is suggested to be involved in a
protein-protein interaction, the carboxyl terminus of synUSP may
participate in a certain type of protein-protein interaction.
synUSP also has two repeats of 6 amino acid residues ((L/I)LCPHG
(SEQ ID NO: 3)) in the carboxyl terminus portion. In the database
search, any information about the function of this sequence was not
obtained.
Example 2
Confirmation of USP Activity of Expression Product
[0056] The activity of synUSP for digesting a model ubiquitinated
protein was investigated by using a co-expression system of E.
coli.
[0057] The USP activity was measured basically following the method
of Everett et al. (EMBO J., 16:1519-1530, 1997) by using a
T7-driven IPTG-inducible synUSP expression plasmid. The pT7-synUSP
plasmid was prepared by inserting the complete coding region of
synUSP into the NcoI site of T7 expression plasmid pET3d (pBR322
Amp.sup.r replicon). pACT7-synUSP which is a plasmid containing a
T7-synUSP expression cassette in pACYC 184 Cm.sup.r replicon was
constructed by inserting an EcoRV fragment of pT7-synUSP at the
EcoRV site of pACYC184. A plasmid pACYC-UBP (PACYC184 Cm.sup.r
replicon) encoding yeast UBP2 and a plasmid pGEX-Ub52 (pBR322
Amp.sup.r replicon) encoding a GST-Ub52 fusion protein were used as
positive controls for USP and a USP substrate. For cleavage of the
GST-Ub52 substrate, cells of the E. coli strain BL21 (DE3)
harboring pGEX-Ub52 were transformed with either pACT7-synUSP or
pACYC-UBP2, and colonies resistant to both of ampicillin and
chloramphenicol were grown-up. The protein expression was induced
by IPTG, and the cells were further incubated for 3 hours to allow
USP to cleave the substrate. The cells were sonicated to prepare an
extract of water-soluble proteins. The GST fusion protein and
proteolysed products thereof were purified by using
glutathione-agarose beads, subjected to SDS-polyacrylamide gel
electrophoresis and detected by staining with Coomassie brilliant
blue. Further, similar procedure was carried out for synUSP in
which Cys98 was replaced with Ala and synUSP of which C-terminus
sequence was deleted from either the His box, LLCPHG repeat
sequence or leucine zipper (FIG. 1,a).
[0058] The results are shown in FIG. 1,b. The expression of
GST-Ub52 substrate protein was induced by IPTG (Lane 1). The
GST-Ub52 protein was degraded to a band of 30 kDa by the
co-expression of yeast USP2. The size of the degradation product
was good agreement with that calculated. Therefore, the validity of
the aforementioned USP activity assay system was confirmed. When a
wild type (WT) synUSP was co-expressed, the amount of the GST-Ub52
protein of 45 kDa decreased, and the proteolysis product of 30 kDa
markedly increased along with it (Lane 3). Therefore, it was
confirmed that synUSP has the USP activity. The activity of synUSP
disappeared by either replacing Cys98 with Ala or deleting the
C-terminus sequence from the His box, LLCPHG repeat sequence or
leucine zipper (Lanes 4 to 7).
Example 3
Preparation of Antibody, Subcellular Distribution and Tissue
Distribution of synUSP
[0059] A peptide (C-15 peptide) consisting of 15 amino acid
residues of the C-terminus of synUSP (amino acid numbers 1022 to
1036 in the amino acid sequence of SEQ ID NO: 2) was coupled to
keyhole limpet hemocyanin and used to immunize a rabbit. After
repeating booster immunization, blood serum was collected and
anti-synUSP antibodies (C-15 Ab) were purified by affinity
chromatography using Affi-Gel 10 on which the C-15 peptide was
immobilized.
[0060] Specificity of the purified C-15 Ab was examined by
performing Western blotting to the full length synUSP expressed in
Cos7 cells according to the method described in J. Biol. Chem.,
276, 21417-21424 (2001). As a result, C-15 Ab specifically reacted
with the expressed synUSP of 125 kDa, and this interaction was
blocked by addition of an excessive amount of the C-15 peptide.
[0061] The subcellular distribution of synUSP was investigated by
using C-15 Ab. A subcellular fraction was obtained as follows. As
described in Mol. Brain Res., 78, 80-90 (2000), fractions of
synaptic plasma membrane (SPM) and PSD were obtained from the
forebrain of Wistar rat (six-week old, male). Further, during the
isolation of PSD, P1 (fraction containing nuclei and cell debris),
P2 (crude mitochondrion fraction) and syn (synaptosome fraction)
were obtained. Furthermore, a soluble fraction and a dendritic
lipid raft fraction were obtained as described in Mol. Brain Res.,
78, 80-90 (2000) and Mol. Brain Res., 89, 20-28 (2001),
respectively.
[0062] Each fraction was analyzed by Westem blotting using C-15 Ab.
As a result, a band of the same molecular weight as the full length
synUSP expressed in the Cos7 cells was detected in the total
homogenate, soluble, dendritic lipid raft and PSD fractions. In the
PSD fraction, immunoreactive bands with different sizes were
detected. However, since the bands disappeared by the addition of
C-15 peptide and the amounts of the bands fluctuated in every
experiment, they appeared to be degradation products of synUSP.
[0063] Western blotting was carried out in the same manner as that
described above for proteins prepared from the heart, brain,
spleen, lung, liver, skeletal muscle, kidney, testis, thymus gland,
stomach and small intestine. As a result, a band of 125 kDa was
detected in the brain and thymus gland, and the expression level in
the thymus gland was lower than that in the brain. Moreover, a band
of the same size was detected also in the testis. However, the
expression level was extremely low. In the brain, immunoreactive
band smaller than the full length was detected. However, since
these bands disappeared by the addition of C-15 peptide and the
amounts of the bands fluctuated in every experiment, they appeared
to be degradation products of synUSP. Further, two immunoreactive
bands were detected in a low molecular weight region also in the
liver. These bands disappeared by the addition of C-15 peptide. No
immunoreactive band was detected in the other tissues.
INDUSTRIAL APPLICABILITY
[0064] According to the present invention, a ubiquitin-specific
protease and a DNA encoding it are provided. These are useful for
researches on neuroplasticity and pathology of neurodegenerative
disorders.
Sequence CWU 1
1
9 1 5738 DNA Artificial Rattus norvegicus misc_feature
(4799)..(4799) n is a, c, g, or t 1 cagctgggcg cgggcgggag
ggtgcggcgg ctgtggcgcg cgcaggtgat tgactggtcc 60 cttggctgag
gggagcgctt ggttctttcg gctggcaggt gccagtgccc gttcgggcgg 120
agacggaggc ggcgccggag gccacccggc tcggcgagtg ctatttcggt caaggccatg
180 gctccgcggc tgcagctaga gaaggcggcc tggcgctggg cggagacggt
gcggcccgag 240 gaggtgtcgc aggagcatat cgagaccgcc taccgcatct
ggctggagcc ctgtatccga 300 ggcgtgtgca gaagaaactg cagaggaaat
ccgaattgct tagttgggat tggcgagcac 360 atttggttag gagaaataga
tgagaatagt tttcataaca ttgatgaccc caactgtgag 420 aggaggaaaa
agaattcgtt tgtgggcctg actaatctgg gagcctcctg ttacgtcaac 480
accttcctcc aggtgtggtt cctcaacttg gagcttcggc aggcgctata cttatgccca
540 agcacctgta gtgattacac gaagggagat ggtatccgtg gaggaaaaga
ttatgagcct 600 cagacaattt gtgagcatct tcagtacttg tttgctttgt
tgcaaaacag taacagacga 660 tacatcgatc cttcaggatt tgttaaagcc
ttgggcttgg acactggcca gcagcaggat 720 gcacaagagt cttccaagct
ctttatgtcg ctgctggaag acactttgtc taaacagaag 780 aacccggatg
ttcggaacgt cgtgcagcag cagttctgtg gggagtacgc ctatgtgaca 840
gtgtgcagtc agtgtggccg agagtctaag ctcgtatcga agttctatga actggagtta
900 aacatccaag gccacaagca gttaacagac tgcatctccg agttcctgaa
ggaagaaagg 960 ttagaaggag acaatcgata cttctgtgaa aactgtcaaa
gcaaacagaa tgccacgcgg 1020 aaaatccgac ttctgagcct cccttgcact
ctgaacttgc agctgatgcg ttttgtgttt 1080 gaccggcaaa ctggacacaa
gaagaaactt aatgcttata ttggcttctc ggagagccta 1140 gatatggagc
catatgtgga gcacaaaggc gggtcctttg tgtacgagct cagcgcggtc 1200
ctcatccaca gaggagtcag tgcctattcc ggccactaca tcgcccacgt gaaagacccc
1260 cagtctggag actggtacaa gttcaatgat gaggacatag agaagatgga
ggggaagaaa 1320 ctacagctag ggattgagga agatctaaca gaaccttcta
agtcccagac ccgaaaaccc 1380 aagtgtggca aaggaactca ctgctctcga
aatgcataca tgttggttta tagactgcaa 1440 acccaggaaa agaaccacac
gatggttcaa gtcccagcct ttctccaaga gctggtggat 1500 cgggataatt
ccaaatttga agagtggtgt gtagaaatgg ccgagatgcg caggcagagt 1560
gtggacaagg gcagagcgaa gcacgaggag gtgaaggagc tgtaccaaag gttgcctgct
1620 ggagctgagc cctatgagtt tgtctctctt gagtggctgc agaagtggct
ggatgaatca 1680 acccctacca aacctattga taataatgcc tgcctgtgtt
cccatgacaa gctccatcca 1740 gacaaaatat caattatgaa gagaatatcg
gaatacgcag ctgacatttt ctatagtcga 1800 tatggaggag gtcccagact
cactgtgaaa gccctctgta aggactgtgt tgtggagcgc 1860 tgtcgcatac
tgcgcctgaa gaatcaacta aatgaagatt acaaaactgt caacaatttg 1920
ctgaaagcaa caatgaaggg cagtgatgga ttttgggtag gaaagtcttc cttgcgcagc
1980 tggcgccagc tggctcttga gcaactagat gagcaagacg gtgaggcgga
gcaaagcaat 2040 ggaaagataa acgggagtcc cttcagtaaa gatgaatcaa
aggaagaaaa gaaggaagag 2100 gaggaggaac taaattttaa tgaagacatt
ctgtgtcctc atggagagtt aagcatatct 2160 gaaaatgaaa gaaggctcgt
ttctcaggag gcttggagca aactgcagca gtatttccca 2220 aaggctcctg
agttcccaag ttacaaagag tgctgctcgc aatgcaagat actagaacga 2280
gaaggtgaag aaaatgaagc cttgcataaa atgattgcaa aggagcaaaa aacctcactt
2340 ccaaacttgt tccaggacaa aaacagaccg tgcctcagta actggccaga
ggacacggac 2400 gctctctata ttgtgtcgca cttcttttta gatgaatggc
ggaaattcgt taggaagcct 2460 gcgaggtcca ctcccgtgtc ctcagttggg
aacgctgccc ttctctgtcc acatggagga 2520 ctcatgttta catttccttc
cctcaccaaa gaagactcga aacttatagc tctcatatgg 2580 cccagcgaat
ggcaaatgat tcaaaagctc tttgttgtgg ataaagtaat taaaatcaca 2640
agaattgaag tgggagacgt aaacccttct caaacacagt atatttctga gcctaatctc
2700 tgtccagatt gcagagaagg tctgctgtgc cagcagcaga aggaccttcg
agagtatacc 2760 caagccacca tctatgtcca caaagttgtg gacaacaaaa
aggtgatgaa ggactcagct 2820 ccagagttga atgtgagtag ttctgagacg
gaggaggaca aggaggaagc taagccagac 2880 ggagagaaag accctgactt
taatcaaagc aatggaggta ccaagcgaca gaaaacatcc 2940 caacagggct
atgtggccta ccagaagcaa gtcatccgga ggagcacgcg gcaccggaag 3000
gttcggggag agaaagcact gctggtgtct gccaaccaga cgttaaaaga actgaagatt
3060 cagatcatgc atgcattctc agttgctccg tttgaccaga atttgtcaat
tgatggaaag 3120 attttaaatg atgactgtgc tacccttggt accctcggtg
tcattcctga gtctgtcatt 3180 ttattaaagg cagatgagcc aatcgcagac
tacgctgcaa tggatgatgt catgcaagtt 3240 tgtatgccgg aagagggctt
taaaggtaca ggcctgcttg gacattaatc tttgaccact 3300 tgttgactgc
caaggaatga ccagaaagga agatgaactt gacctgttag ggcattaaag 3360
aaaatgtgga tttaagaatt aaactcttac ctggctcttc caaaaggcaa aaatccattc
3420 aaagagactg tcccaattgc cttatgtcaa ataaagcaga ttgcactggt
ggacatcaga 3480 cttgaaggaa atgtttcagg ttttctattt agggtgggag
ggagagggcg agagaggact 3540 gagcaggcag agtaaatcat gttaacacag
gagggcccaa agtccttttc aagtttcaca 3600 cagatgtcct ggtcagtagt
ggatttgtca cagtacatgg gctagcatcc tgctccaaac 3660 tctcaagtgg
cttgtctaga gacgcgacct gctaacccac cctgtctttg acctgtactc 3720
atcttccaat aaagcatgaa agtgaaggac ttgccaacaa ggaacgggtc ttcagagcgc
3780 cactcccgca ggacagccac agccctgcgg cctgctccgt gtctgtcagt
tctgggttga 3840 aagcacccgt gtgctttcct tgtcaagcca gggaccgtct
cagtttatct ccatcattcc 3900 agaggtgatg ggaaatgttc agcaaagaaa
gtatccctga acatttgaaa agccggacag 3960 aagagtgacc gcttgcctga
agctccgtag gacagcacgg gtttttatag acaaccctcc 4020 cattctccag
ttcctctttc ctcggacctc acttctttct gaaaaccatt cttctgatac 4080
atcctacagg ggcatgttga aaggaggaac aaaagactat acagggcgtg ctgtctctcc
4140 agctcaggat cacattgatt agtctaagca tcaggatctg caggaacaga
aaggctttag 4200 cagagcatgt ggctgggctg ggcagatttg cattgtctgt
cgagtggatg ttggtatgtt 4260 tgtggcccag ttgctcacac agattttttt
gttgttgtta cttttttcct tttaaatctc 4320 tagtttaaaa accttaactg
atgtgtacaa gctgatctca acattagcag catcttttga 4380 tagctttctg
ccagtgctat ggctgctggg taaaactctg attgtggtgt tttaggattg 4440
caatgaagag tgttctctag gtggtgagca gcgcagagca gagccgggag agctgcacac
4500 cgtcctggag gagtactcac ggagctcctg cctttgggga cacagggact
caggtgacac 4560 tagttttcag gtctgttctt tactgagcac cacagatatg
ggaccaagcc tcactcacca 4620 agagcctact ctttcccaag gccaagacaa
ttgaaaggtc gccaaatatt tggacaagtg 4680 acctttattg tccttacaaa
gaaacccgga gcttgaggct gtgtgacaaa gcgttggtag 4740 ggacctctgc
acccctaaaa ggtgctgggg ataagtggta aaaagctgtg tgcaccccnt 4800
cccccgtccc ccttcttggg attgtaagtg cacaagccag aagttagcac tgtgagattg
4860 tttgcatagg catcttttgt ttcctttgca taatattagc aaagtaatag
ccattgactc 4920 ttgtcaggca aatgcaggtg gggcagaccc agtgtcagca
acccagtgtc tgccgcttcc 4980 gcctcgacat cagtcgcccc atcgcccact
gcagagcctt attgggaact tcagctcaga 5040 ccccgcaccc accgaacacg
gccactcccg aaaagcttgg gacccctggg ctgcggcctc 5100 ctcacagctt
gaagcagaca aatttccgag tatccacgtg gacttagcag ctgccatggc 5160
cctgctttct aatccgtgct gtttggttgt cttctgaact gtggtggctc tacagcacag
5220 gctagctctt ggttctaaag ccacatccag aaaccgcact aaaccagatt
atgaatctcc 5280 aggcttcttt atgtttcttt tgtctttccc agacaggtct
cctgtaaccc aggctggcct 5340 cagactcact atgtaaccag gataacctta
acttcccatc ttcctacttc ctcctcccag 5400 gtgctgggat tgcaggctcg
tggccactgt gacaggtttc ctgtgctgtg tggaggcccg 5460 agcccatggt
ttcgtgcaca ctagacaagc gttctgcaca ctgacccatg tccccagctc 5520
tacccatgta tatttctaat cattctttaa tcccaagcaa acgtggagtc tcctatgctg
5580 agtccattac cagactgtag gttatcattt gatgtcttta ttccctgaga
gaaacctctt 5640 ctgtatttat gaacttgatt attgagaatc actgttaatc
tttctgccaa attaaagaca 5700 cccatgtctg tttccacgga aaaaaaaaaa
aaaaaaaa 5738 2 1036 PRT Artificial Rattus norvegicus 2 Met Ala Pro
Arg Leu Gln Leu Glu Lys Ala Ala Trp Arg Trp Ala Glu 1 5 10 15 Thr
Val Arg Pro Glu Glu Val Ser Gln Glu His Ile Glu Thr Ala Tyr 20 25
30 Arg Ile Trp Leu Glu Pro Cys Ile Arg Gly Val Cys Arg Arg Asn Cys
35 40 45 Arg Gly Asn Pro Asn Cys Leu Val Gly Ile Gly Glu His Ile
Trp Leu 50 55 60 Gly Glu Ile Asp Glu Asn Ser Phe His Asn Ile Asp
Asp Pro Asn Cys 65 70 75 80 Glu Arg Arg Lys Lys Asn Ser Phe Val Gly
Leu Thr Asn Leu Gly Ala 85 90 95 Ser Cys Tyr Val Asn Thr Phe Leu
Gln Val Trp Phe Leu Asn Leu Glu 100 105 110 Leu Arg Gln Ala Leu Tyr
Leu Cys Pro Ser Thr Cys Ser Asp Tyr Thr 115 120 125 Lys Gly Asp Gly
Ile Arg Gly Gly Lys Asp Tyr Glu Pro Gln Thr Ile 130 135 140 Cys Glu
His Leu Gln Tyr Leu Phe Ala Leu Leu Gln Asn Ser Asn Arg 145 150 155
160 Arg Tyr Ile Asp Pro Ser Gly Phe Val Lys Ala Leu Gly Leu Asp Thr
165 170 175 Gly Gln Gln Gln Asp Ala Gln Glu Ser Ser Lys Leu Phe Met
Ser Leu 180 185 190 Leu Glu Asp Thr Leu Ser Lys Gln Lys Asn Pro Asp
Val Arg Asn Val 195 200 205 Val Gln Gln Gln Phe Cys Gly Glu Tyr Ala
Tyr Val Thr Val Cys Ser 210 215 220 Gln Cys Gly Arg Glu Ser Lys Leu
Val Ser Lys Phe Tyr Glu Leu Glu 225 230 235 240 Leu Asn Ile Gln Gly
His Lys Gln Leu Thr Asp Cys Ile Ser Glu Phe 245 250 255 Leu Lys Glu
Glu Arg Leu Glu Gly Asp Asn Arg Tyr Phe Cys Glu Asn 260 265 270 Cys
Gln Ser Lys Gln Asn Ala Thr Arg Lys Ile Arg Leu Leu Ser Leu 275 280
285 Pro Cys Thr Leu Asn Leu Gln Leu Met Arg Phe Val Phe Asp Arg Gln
290 295 300 Thr Gly His Lys Lys Lys Leu Asn Ala Tyr Ile Gly Phe Ser
Glu Ser 305 310 315 320 Leu Asp Met Glu Pro Tyr Val Glu His Lys Gly
Gly Ser Phe Val Tyr 325 330 335 Glu Leu Ser Ala Val Leu Ile His Arg
Gly Val Ser Ala Tyr Ser Gly 340 345 350 His Tyr Ile Ala His Val Lys
Asp Pro Gln Ser Gly Asp Trp Tyr Lys 355 360 365 Phe Asn Asp Glu Asp
Ile Glu Lys Met Glu Gly Lys Lys Leu Gln Leu 370 375 380 Gly Ile Glu
Glu Asp Leu Thr Glu Pro Ser Lys Ser Gln Thr Arg Lys 385 390 395 400
Pro Lys Cys Gly Lys Gly Thr His Cys Ser Arg Asn Ala Tyr Met Leu 405
410 415 Val Tyr Arg Leu Gln Thr Gln Glu Lys Asn His Thr Met Val Gln
Val 420 425 430 Pro Ala Phe Leu Gln Glu Leu Val Asp Arg Asp Asn Ser
Lys Phe Glu 435 440 445 Glu Trp Cys Val Glu Met Ala Glu Met Arg Arg
Gln Ser Val Asp Lys 450 455 460 Gly Arg Ala Lys His Glu Glu Val Lys
Glu Leu Tyr Gln Arg Leu Pro 465 470 475 480 Ala Gly Ala Glu Pro Tyr
Glu Phe Val Ser Leu Glu Trp Leu Gln Lys 485 490 495 Trp Leu Asp Glu
Ser Thr Pro Thr Lys Pro Ile Asp Asn Asn Ala Cys 500 505 510 Leu Cys
Ser His Asp Lys Leu His Pro Asp Lys Ile Ser Ile Met Lys 515 520 525
Arg Ile Ser Glu Tyr Ala Ala Asp Ile Phe Tyr Ser Arg Tyr Gly Gly 530
535 540 Gly Pro Arg Leu Thr Val Lys Ala Leu Cys Lys Asp Cys Val Val
Glu 545 550 555 560 Arg Cys Arg Ile Leu Arg Leu Lys Asn Gln Leu Asn
Glu Asp Tyr Lys 565 570 575 Thr Val Asn Asn Leu Leu Lys Ala Thr Met
Lys Gly Ser Asp Gly Phe 580 585 590 Trp Val Gly Lys Ser Ser Leu Arg
Ser Trp Arg Gln Leu Ala Leu Glu 595 600 605 Gln Leu Asp Glu Gln Asp
Gly Glu Ala Glu Gln Ser Asn Gly Lys Ile 610 615 620 Asn Gly Ser Pro
Phe Ser Lys Asp Glu Ser Lys Glu Glu Lys Lys Glu 625 630 635 640 Glu
Glu Glu Glu Leu Asn Phe Asn Glu Asp Ile Leu Cys Pro His Gly 645 650
655 Glu Leu Ser Ile Ser Glu Asn Glu Arg Arg Leu Val Ser Gln Glu Ala
660 665 670 Trp Ser Lys Leu Gln Gln Tyr Phe Pro Lys Ala Pro Glu Phe
Pro Ser 675 680 685 Tyr Lys Glu Cys Cys Ser Gln Cys Lys Ile Leu Glu
Arg Glu Gly Glu 690 695 700 Glu Asn Glu Ala Leu His Lys Met Ile Ala
Lys Glu Gln Lys Thr Ser 705 710 715 720 Leu Pro Asn Leu Phe Gln Asp
Lys Asn Arg Pro Cys Leu Ser Asn Trp 725 730 735 Pro Glu Asp Thr Asp
Ala Leu Tyr Ile Val Ser His Phe Phe Leu Asp 740 745 750 Glu Trp Arg
Lys Phe Val Arg Lys Pro Ala Arg Ser Thr Pro Val Ser 755 760 765 Ser
Val Gly Asn Ala Ala Leu Leu Cys Pro His Gly Gly Leu Met Phe 770 775
780 Thr Phe Pro Ser Leu Thr Lys Glu Asp Ser Lys Leu Ile Ala Leu Ile
785 790 795 800 Trp Pro Ser Glu Trp Gln Met Ile Gln Lys Leu Phe Val
Val Asp Lys 805 810 815 Val Ile Lys Ile Thr Arg Ile Glu Val Gly Asp
Val Asn Pro Ser Gln 820 825 830 Thr Gln Tyr Ile Ser Glu Pro Asn Leu
Cys Pro Asp Cys Arg Glu Gly 835 840 845 Leu Leu Cys Gln Gln Gln Lys
Asp Leu Arg Glu Tyr Thr Gln Ala Thr 850 855 860 Ile Tyr Val His Lys
Val Val Asp Asn Lys Lys Val Met Lys Asp Ser 865 870 875 880 Ala Pro
Glu Leu Asn Val Ser Ser Ser Glu Thr Glu Glu Asp Lys Glu 885 890 895
Glu Ala Lys Pro Asp Gly Glu Lys Asp Pro Asp Phe Asn Gln Ser Asn 900
905 910 Gly Gly Thr Lys Arg Gln Lys Thr Ser Gln Gln Gly Tyr Val Ala
Tyr 915 920 925 Gln Lys Gln Val Ile Arg Arg Ser Thr Arg His Arg Lys
Val Arg Gly 930 935 940 Glu Lys Ala Leu Leu Val Ser Ala Asn Gln Thr
Leu Lys Glu Leu Lys 945 950 955 960 Ile Gln Ile Met His Ala Phe Ser
Val Ala Pro Phe Asp Gln Asn Leu 965 970 975 Ser Ile Asp Gly Lys Ile
Leu Asn Asp Asp Cys Ala Thr Leu Gly Thr 980 985 990 Leu Gly Val Ile
Pro Glu Ser Val Ile Leu Leu Lys Ala Asp Glu Pro 995 1000 1005 Ile
Ala Asp Tyr Ala Ala Met Asp Asp Val Met Gln Val Cys Met 1010 1015
1020 Pro Glu Glu Gly Phe Lys Gly Thr Gly Leu Leu Gly His 1025 1030
1035 3 6 PRT Rattus norvegicus misc_feature (1)..(1) Xaa can be any
naturally occurring amino acid 3 Xaa Leu Cys Pro His Gly 1 5 4 14
DNA Artificial RT Primer 4 accagctctt ggag 14 5 24 DNA Artificial
antisense primer 5 tagaacttcg atacgagctt agac 24 6 21 DNA
Artificial sense primer 6 gtcagtgcct attccggcca c 21 7 12 DNA
Artificial RT primer 7 cagattagtc ag 12 8 23 DNA Artificial
antisense primer 8 ccatggcctt gaccgaaata gca 23 9 18 DNA Artificial
sense primer 9 gtcgcaggag catatcga 18
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References